A key finding of these more recent stutdies is that, after SIV challenge, the anamnestic SIVspecific CD8+ T cells responses in SHIV-immunized monkeys were limited to the vaginal mucosa and there was minimal immune activation after the SIV challenge. Importantly, the magnitude of the CD8+ T cell responses in the vagina of SHIV-immunized animals (100-500 SIVgag-specific T cells/10^ CD3+ T cells) was not appreciably greater than T cell responses elicited in PBMC of HIV vaccine clinical trial participants by numerous candidate HIV vaccines. Thus it is difficult to explain the consistent protection in this model based only on the strength of the SHIV-elicited CD8+ T cells response. Clearly the anatomic location of the CD8+ T response in the vaginal mucosa may be an explanation for the SHIV-mediated protection, but that does not seem to be a sufficient to explain the consistent protection observed in the model. A striking observation from the analysis of immune responses in tissues of SHIV-immunized animals was that there is relatively quiescent environment in the genital tract mucosa in which the CD8+ T cells encounter the virus. In fact we found that, in contrast to unimmunized control animals, Treg cells and associated regulatory genes are elevated and maintained in the vaginal mucosa of SHIV-immunized animals within 3-14 days of SIV challenge. We now believe that both a modest CD8+ T cell response in the vaginal mucosa and minimal immune activation in the vaginal mucosa are NECESSARY for protection in this model system. Thus in this project, we will directly and indirectly test the hypothesis that was generated as a result of initial funding period. Finally, we intend to determine the extent to which SHIVimmunization can protect animals with pre-existing genital tract inflammation from vaginal SIV challenge and to determine if SHIV-immunization can elicit protection against repeated low dose vaginal and penile SIV exposure. These latter 2 AIMS will realistically model the conditions of HIV transmission and provide a rigorous test of the limits of SHIV-mediated protection.